Infact, this could lead to a separation from the second peptide as this
might stick to the cat-ex column. Further purification could be done with
the material that did not bind by means of reversed-phase.
Michael Schrader
----------------------------------------
BioVisioN GmbH & Co. KG
Feodor-Lynen-Str. 5
D-30625 Hannover
GERMANY
fon +49 (0)511-53 88 96-12
fax +49 (0)511-53 88 96-66
Internet www.biovision.de
E-mail m.schrader@biovision.de
> -----Urspržngliche Nachricht-----
> Von: POLYLC@aol.com [SMTP:POLYLC@aol.com]
> Gesendet am: Mittwoch, 1. Dezember 1999 17:24
> An: M.Schrader@biovision.de
> Cc: abrf-request@aecom.yu.edu; abrf@aecom.yu.edu;
> jennys@biols.susx.ac.uk
> Betreff: Re: Re[2]: IE HPLC
>
> Hi, Michael!
>
> Pardon my skepticism, but it will be hard to get adequate retention of a
> 30-residue peptide if the only basic group is the N-terminus (assuming it
> is
> unblocked). At best, you'll get elution about 3-4 column volumes past the
>
> void when eluting isocratically with a low-salt buffer (e.g., 5 mM K-PO4,
> pH
> 2.7, with 20% ACN). Jenny stands a better chance by using 50 mM K-PO4, pH
>
> 2.0, with 10% ACN, to elute her peptide from the anion-exchange column.
> These conditions will uncharge the Glu- residues.
>
> One possibility may be that her Glu- residues are in close proximity and
> that
> they're chelating heavy metals in the frits. If this is the case, then
> she
> can passivate her column by eluting 24 hours with 40 mM EDTA.2Na.
>
> Best regards,
>
> Andy Alpert
> PolyLC Inc.
> 9151 Rumsey Road, ste. 180
> Columbia, MD 21045 USA
> tel: (410) 992-5400 FAX: (410) 730-8340
> *********************************************************************
> << Subj: Re[2]: IE HPLC
> Date: 12/01/1999 9:14:17 AM Eastern Standard Time
> From: M.Schrader@biovision.de (Schrader, Michael)
> Sender: abrf-request@aecom.yu.edu (Association of Biomolecular
> Resource
> Facilities)
> To: abrf@aecom.yu.edu (Recipients of ABRF List)
>
> Dear Jenny
>
> We have very good experience with cation-exchange chromatography for
> peptides and I would suggest to try that. Your peptide won't be sticky
> then.
> Try strong exchanger pH around 3, usually you get very sharp peaks.
>
> Michael Schrader
> ----------------------------------------
> BioVisioN GmbH & Co. KG
> Feodor-Lynen-Str. 5
> D-30625 Hannover
> GERMANY
> fon +49 (0)511-53 88 96-12
> fax +49 (0)511-53 88 96-66
> Internet www.biovision.de
> E-mail m.schrader@biovision.de
>
> > -----Urspržngliche Nachricht-----
> > Von: Jenny Shipway [SMTP:jennys@biols.susx.ac.uk]
> > Gesendet am: Dienstag, 30. November 1999 18:26
> > An: Recipients of ABRF List
> > Betreff: IE HPLC
> >
> >
> > Hello you ABRFers,
> >
> > Help!
> >
> > It's easy, I thought, just get an anion-exchange column (DEAE) and get
> a
> > salt gradient going. Hah!
> >
> > I have a highly charged peptide (30aa, 4 Glu's, no positive charges)
> which
> > is refusing to budge from the column. I've been running 10mM Tris (pH
> 8.5
> > - I want to seperate this guy from another with pI about 7, and didn't
> > want to do a pH gradient) and shoving loads of NaCl in, but nothing
> comes
> > off. I just ran 2M NaCl for 1.5h (this is how Vydac say I should clean
> the
> > column of any highly charged stuff) and still nothing.
> >
> > Is there anything I can do to avoid doing a pH gradient? (Kosmotropic
> > salts?) I don't want to fill the entire HPLC system with salt crystals.
> > This negative peptide has a pI of about 3, so what buffer systems would
> be
> > good for going from pH8 to pH3? Is it okay doing a pH gradient with
> > DEAE? (I read somewhere that it's much better to do salt gradients(?))
> >
> > Any thoughts would be appreciated!
> >
> > Jenny Shipway
> > Centre for Biomolecular Design and Drug Development
> > University of Sussex